Combined ultrahigh-resolution optical tweezers and single-molecule fluorescence

描述(由申请人提供)：单分子技术已经发展成为研究许多基本生物过程中涉及的分子机器的强大工具。荧光定位、F“rster共振能量转移(FRET)和光钳等技术在破译肌球蛋白和动蛋白、DNA和RNA聚合酶以及解旋酶等分子马达的机制方面发挥了重要作用，仅举几个例子。最近，超高分辨率光学镊子的发展首次使直接观察分子运动成为可能，其尺度为1碱基DNA(3.4°)。尽管取得了这些进展，单分子技术还是有很大的局限性。尽管分子机器中涉及的构象变化本质上是三维的，但这种技术通常将所有运动投射到一个轴上，因此不能捕捉分子运动的全部复杂性。此外，这些技术在很大程度上局限于简单的系统，只涉及很少的成分被分离检查，而在细胞环境中，分子机器由高度协调的多组分蛋白质组装组成。 为了解决这些局限性，我们建议1)开发结合多色荧光检测和超高分辨率光钳的新一代单分子仪器。尽管之前已经开发了融合荧光和光学陷阱的仪器，但实现Basepair分辨率仍然是一个巨大的挑战，需要一种新的方法。然而，这些能力对于理解参与DNA新陈代谢的分子复合体--转录、复制、重组和修复--是至关重要的，这些分子复合体具有重大的生物医学意义。我们提出的混合仪器将能够同时测量多个可观测对象，例如通过FRET测量内部蛋白质动力学或通过荧光定位测量蛋白质复合体的组装动力学，并结合使用光学镊子在基本空间分辨率下检测马达位移。作为这项技术的演示，我们将2)在碱基分辨下监测大肠杆菌Rep解旋酶的易位和双链解离，同时通过FRET监测构象变化，通过荧光检测监测寡聚体状态。这项拟议的工作涉及伊利诺伊大学厄巴纳-香槟分校的超高分辨率光学镊子(Y.Chemla，Pi)和单分子荧光(T.Ha，co-Pi)专家的合作。 公共卫生相关声明：我们正在提议开发一种仪器，它将结合两种强大的尖端技术：单分子荧光和超高分辨率光学捕获。我们的目标是在ngstrom水平上研究参与DNA复制、转录、重组和修复的蛋白质和蛋白质复合体的动力学。这项拟议的技术有可能揭示这些分子机器的详细机制，这是一个非常有医学意义的问题，因为它们的活动缺陷与许多人类疾病有关，特别是癌症。

DESCRIPTION (provided by applicant): Single molecule techniques have developed into a powerful tool to study the molecular machines involved in many fundamental biological processes. Techniques such as fluorescence localization, F"rster resonance energy transfer (FRET), and optical tweezers have been instrumental in deciphering the mechanism of molecular motors such as myosin and kinesin, DNA and RNA polymerases, and helicases, to name just a few examples. Recently, the development of ultrahigh-resolution optical tweezers has made possible, for the first time, the direct observation of molecular motion on the scale of 1 basepair of DNA (3.4¿). Despite such advances, single molecule techniques have had important limitations. Although the conformation changes involved in molecular machines are inherently three-dimensional, such techniques typically project all motion onto a single axis and thus cannot capture the full complexity of molecular motion. Furthermore, these techniques have largely been limited to simple systems involving very few components examined in isolation, whereas, in the cellular context, molecular machines consist of highly coordinated multi-component protein assemblies. To address these limitations, we propose to 1) develop the new generation of single molecule instrumentation combining multi-color fluorescence detection and ultrahigh-resolution optical tweezers. Although instruments merging fluorescence and optical traps have been developed previously, achieving basepair resolution remains a grand challenge that will require a new approach. These capabilities nevertheless will be essential to understand the molecular complexes involved in DNA metabolism-transcription, replication, recombination, and repair-that have great biomedical significance. The hybrid instrument we propose will have the ability to measure multiple observables simultaneously, such as internal protein dynamics by FRET or the assembly kinetics of protein complexes by fluorescence localization, combined with detection of motor displacement at basepair resolution by optical tweezers. As a demonstration of this technique, we will 2) monitor translocation and duplex unwinding by E. coli Rep helicase at basepair resolution, simultaneously with conformational changes by FRET and oligomeric state by fluorescence detection. This proposed work involves the collaboration of experts in ultrahigh-resolution optical tweezers (Y. Chemla, PI) and single-molecule fluorescence (T. Ha, co-PI) at the University of Illinois, Urbana-Champaign. Public Health Relevance Statement: We are proposing to develop an instrument that will combine two powerful cutting-edge technologies: single-molecule fluorescence and ultrahigh-resolution optical trapping. Our goal is to study the dynamics of proteins and protein complexes involved in DNA replication, transcription, recombination, and repair at ¿ngstrom level resolution. This proposed technique has the potential to reveal the detailed mechanism of these molecular machines, a problem of great medical interest as defects in their activity have been implicated in a number of human diseases, specifically cancer.